It is frequently necessary to combine reagents in a biochemical or chemical reaction. Typically, biological reagents such as proteins and nucleic acid polymers are delivered from aqueous solutions or suspensions or from freeze-dried material.
There are many problems associated with using aqueous solutions for reagent delivery. Proteins in an aqueous solution or suspension may be subject to rapid decomposition and loss of activity. Therefore, aqueous solutions or suspensions must usually be stored at a low temperature. Additionally, it is difficult to combine an aqueous reagent solution with a general reaction mixture without the reagents reacting immediately. Pipetting and dispensing aqueous reagents can be subject to measurement errors. It is also difficult to automate the delivery of aqueous reagents. There is always a potential for cross-contamination when aqueous reagents are repetitively pipetted from mixture to mixture.
Another method for storage and delivery of biological reagents involves drying the reagent. However, freeze-drying and other drying methods must rely on special drying and storage processes to stabilize and deliver reagents for molecular manipulations. For example, WO 87/00196 describes a method for the preservation of biological samples by drying in the presence of trehalose. U.S. Pat. No. 5,098,893 describes storage of material in a glassy or rubbery composition consisting of water-soluble or water-swellable carbohydrates and derivatives. EPA 0298669 describes an apparatus and method for performing nucleic acid manipulations utilizing predetermined amounts of reagents in a dry state. WO 84/03715 and WO 84/03444 describe a method in which reagents and antibodies may be provided in lyophilized form. Expensive and cumbersome methods and equipment, such as vacuum ovens, are required to preserve and deliver dried reagents.
What is needed in the art of molecular biology is an improved method of storing and sequentially delivering reagents in a biochemical or chemical reaction mixture. The present invention relates to a method of storing and sequentially delivering reagents by combining at least one of the reagents with a wax carrier material.
Clinical tissue samples have been stored in paraffin to preserve biologically inactive material for clinical sectioning and subsequent microscopic visualization. DNA has been extracted for PCR amplification from tissue samples prepared for routine histopathological examinations (Goelz, et al., Biochem. Biophys. Res. Commun. 130:118-126, 1985). RNA has been extracted and PCR-amplified from paraffin-embedded human tissue samples (Stanta and Schneider, BioTechniques. 11:304-308, 1991).
Wax has been used as an addition to enzymatic reactions. For example, a recent refinement in the polymerase chain reaction (PCR) process has been the use of AMPLIWAX (Perkin Elmer Cetus) and the development of the "Hot Start" technique. These refinements involve the replacement of mineral oil with a wax pellet as a vapor barrier and to increase amplification specificity (Chou, al., Nucl. Acids Res. 20 [7] 1717-1723, 1992). WO 91/12342 discloses the use of waxes and greases as vapor barriers in PCR reactions. Additionally, WO 91/12342 discloses subsets of PCR reagents, such as Mg.sup.+2, as aqueous emulsions in a grease or wax barrier. European Patent Application 0572057 discloses PCR reagents in inert, meltable materials.